General flow cytometry protocol

Here we provide you with general information about the application of antibodies for flow cytometry (FACS analysis):

Cells

Cells should be kept on ice until use. Make sure that the cell suspension does not contain media components since it could influence the results (e.g. phenolred or other colors). Hence, wash the cells thoroughly before application.

Amount of antibody

It is advised to follow the manufacturers instructions. It is recommended to perform optimization experiments beforehand, in order to determine the optimal amount of antibody. In order to do so, color the cells with varying amounts of antibody in order to determine the lowest possible concentration that results in only a minimal surplus of antibody.

Storage of cells

Optimal results are obtained by measuring the cells immediately after labeling. Alternatively, the cells can be stored refrigerated for some hours. Longer storage (e.g. over night) requires stabilization with 1% parafomaldehyde.

Buffer

If possible, only apply ice-cold buffers and reagents. Use PBS without Mg2+ und Ca2+. Low temperatures and addition of sodium azide prevent modification or internalization.

Dissolved Antibodies

Centrifuge the antibody-suspension briefly before adding to the tubes. Observe, that droplets often remain on the lid or the walls of the vessel. Dissolved antibodies should be keep dark and cold. FACS-tubes with added antibodies must also be stored in the fridge. Flurochromes coupled to antibodies are sensitive to light.

Control experiments

A must-have for optimal results are controls for all colors used. In case of multi-colorings use one control-probe for per individual color and isotype. These controls are then used for adjustment of the FACS-machines, and in order to avoid false positive cells (unspecific binding of Fc-receptors; isotope control).

Hint: antibodies-online.com offers more than for flow cytometry (FACS analysis).